U.S. patent number 7,358,098 [Application Number 11/195,701] was granted by the patent office on 2008-04-15 for device for capturing beads and method and apparatus for arraying beads.
This patent grant is currently assigned to Hitachi Software Engineering Co., Ltd.. Invention is credited to Yoshinobu Kohara, Hideyuki Noda.
United States Patent |
7,358,098 |
Noda , et al. |
April 15, 2008 |
Device for capturing beads and method and apparatus for arraying
beads
Abstract
Beads having diameters of several tens of micrometers to several
millimeters and immobilized with biomolecules are captured by one
kind of bead capturing nozzle one by one without fail. Using a bead
holding plate having a plurality of wells each of which holds a
plurality of the beads and a solution, a vibration generator
mounted with a stage to attach the bead holding plate, and a bead
capturing nozzle for bead capture connected to a suction pump, only
a single bead is captured at the tip of the bead capturing nozzle
by inserting the nozzle having an inner diameter smaller than the
beads and a negative pressure inside created by the pump into the
solution to allow the nozzle to come in contact with the beads in
the well, applying a vibration to the bead holding plate by the
vibration generator, and withdrawing the bead capturing nozzle into
the air.
Inventors: |
Noda; Hideyuki (Kokubunji,
JP), Kohara; Yoshinobu (Mitaka, JP) |
Assignee: |
Hitachi Software Engineering Co.,
Ltd. (Kanagawa, JP)
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Family
ID: |
36603437 |
Appl.
No.: |
11/195,701 |
Filed: |
August 3, 2005 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060257994 A1 |
Nov 16, 2006 |
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Foreign Application Priority Data
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May 13, 2005 [JP] |
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2005-141569 |
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Current U.S.
Class: |
436/518 |
Current CPC
Class: |
B01J
19/0046 (20130101); B01L 3/021 (20130101); G01N
35/10 (20130101); B01J 2219/00315 (20130101); B01J
2219/00364 (20130101); B01J 2219/00373 (20130101); B01J
2219/00414 (20130101); B01J 2219/00466 (20130101); B01J
2219/00468 (20130101); B01J 2219/005 (20130101); B01J
2219/0052 (20130101); B01L 2200/0657 (20130101); B01L
2400/049 (20130101); G01N 2035/00574 (20130101) |
Current International
Class: |
G01N
33/543 (20060101) |
Field of
Search: |
;436/518
;435/283.1,286.7-287.3,288.4 ;422/58,62-66 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1 522 340 |
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Sep 2004 |
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EP |
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11-243997 |
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Mar 1998 |
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JP |
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2000-346842 |
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Apr 2000 |
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JP |
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2003-315336 |
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Apr 2002 |
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JP |
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2005-017224 |
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Jun 2003 |
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JP |
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Other References
H Noda et al., "Automated Bead Alignment Apparatus Using a Single
Bead Capturing Technique for Fabrication of a Miniaturized
Bead-Based DNA Probe Array", Anal. Chem., vol. 75, No. 13 (Jul. 1,
2003), pp. 3250-3255. cited by other .
S. Fodor et al., "Light-Directed,Spatially Addressable Parallel
Chemical Synthesis", Science, vol. 251 (Feb. 15, 1991), pp.
767-773. cited by other .
M. Schena et al., "Quantitative Monitoring of Gene Expression
Patterns with a Complementary DNA Microarray", Science, vol. 270
(Oct. 20, 1995), pp. 467-470. cited by other .
T. Okamoto et al., "Microarray Fabrication with Covalent Attachment
of DNA Using Bubble Jet Technology", Nature Biotechnology, vol. 18
(Apr. 2000), pp. 438-441. cited by other .
R. J. Fulton et al., "Advanced Multiplexed Analysis with the
FlowMetrix.TM. System", Clinical Chemistry, vol. 43, No. 9 (1997),
pp. 1749-1756. cited by other .
Y. Kohara et al., "DNA Probes on Beads Arrayed in a Capillary,
`Bead-array`, Exhibited High Hybridization Performance", Nucleic
Acids Research (2002), vol. 30, No. 16, pp. 1-7. cited by
other.
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Primary Examiner: Lam; Ann Y.
Attorney, Agent or Firm: Reed Smith LLP Fisher, Esq.;
Stanley P. Marquez, Esq.; Juan Carlos A.
Claims
What is claimed is:
1. A device for capturing beads comprising: a chamber to hold a
solution containing a plurality of beads; a vibration generator to
vibrate the chamber; a long and narrow bead capturing nozzle to
capture the beads at a tip thereof; a suction pump connected to the
bead capturing nozzle; and an actuator to insert the tip of the
bead capturing nozzle into the solution in the chamber as well as
to lift up the bead capturing nozzle, wherein an opening at the tip
of the bead capturing nozzle is formed as smaller than a diameter
of several tens of micrometers to several millimeters of each of
the beads, and an outer diameter of the tip of the bead capturing
nozzle is larger than the diameter of several tens of micrometers
to several millimeters of each of the beads.
2. The device for capturing beads according to claim 1, wherein the
vibration generator generates a vibration of a frequency equal to
or higher than 20 Hz.
3. The device for capturing beads according to claim 2, wherein the
amplitude of the vibration generated by the vibration generator is
equal to or larger than 0.1 mm.
4. The device for capturing beads according to claim 1, wherein the
vibration generator vibrates the chamber in the direction
perpendicular to the axis direction of the bead capturing
nozzle.
5. The device for capturing beads according to claim 1, wherein the
vibration generator generates vibration such that the center of the
chamber is rotated in a plane perpendicular to the axis direction
of the bead capturing nozzle.
6. A method for arraying beads comprising the steps of: inserting,
into a solution in a chamber containing a plurality of beads
immobilized with a biomolecular probe on the surface thereof, a
bead capturing nozzle having an opening smaller than the diameter
of the beads formed at the tip thereof and the outer diameter of
the tip larger than the diameter of the beads; exerting a suction
force on the tip of the bead capturing nozzle; vibrating the
chamber; withdrawing the bead capturing nozzle retaining a single
bead at the tip via suction from the solution in the chamber; and
introducing the bead retained at the tip of the bead capturing
nozzle via suction into a bead array container.
7. The method for arraying beads according to claim 6, wherein a
plurality of kinds of beads immobilized with different biomolecular
probes are arrayed in a row in the bead array container in orderly
sequence.
8. The method for arraying beads according to claim 6, wherein the
bead array container is provided with a plurality of flow channels
having different inner diameters that are formed on a same chip and
beads having different diameters corresponding to each inner
diameter are introduced into the plurality of flow channels,
respectively.
9. The method for arraying beads according to claim 6, wherein a
vibration having a frequency equal to or higher than 20 Hz is
provided to the chamber at the step of vibrating the chamber.
10. The method for arraying beads according to claim 6, wherein the
amplitude of the vibration is equal to or larger than 0.1 mm.
11. An apparatus for arraying beads comprising: a stage to retain a
plurality of chambers to hold solutions containing a plurality of
beads respectively; a bead capturing nozzle having an opening
smaller than the diameter of the beads formed at the tip thereof
and an outer diameter of the tip larger than the diameter of the
beads; an actuator to drive the bead capturing nozzle; a
vacuum/pressure unit to develop a negative or positive pressure at
the tip of the bead capturing nozzle; a vibration unit to vibrate
the stage; a holder for bead array container that retains a bead
array container to hold beads; and a control unit, wherein the
control unit allows the bead capturing nozzle to be inserted into
the vibrating chamber on the stage by controlling the actuator, the
bead capturing nozzle to be lifted up from the chamber by
controlling the actuator in a state that a single bead is captured
at the tip of the bead capturing nozzle by controlling the
vacuum/pressure unit to develop a negative pressure at the tip of
the bead capturing nozzle, the bead capturing nozzle capturing a
single bead at the tip to be moved to the position of the bead
array container by controlling the actuator, and the captured bead
to be introduced into the bead array container by controlling the
vacuum/pressure unit to develop a positive pressure at the tip.
12. The apparatus for arraying beads according to claim 11, wherein
a plurality of the bead capturing nozzles are arranged and the
holder for bead array container retains the same number of the bead
array containers as the number of the bead capturing nozzles.
13. The apparatus for arraying beads according to claim 11, wherein
a unit to detect the state that the bead is retained at the tip of
the bead retaining nozzle is provided.
14. The apparatus for arraying beads according to claim 11, wherein
a unit to detect the state that the bead is introduced into the
bead array container is provided.
15. The apparatus for arraying beads according to claim 11, wherein
the holder for bead array container retains the bead array
container provided with a plurality of flow channels with different
inner diameters on a same chip.
16. The device for capturing beads according to claim 1, wherein
each of the beads has biomolecular probes or protein immobilized
thereon.
17. A device for capturing beads comprising: a chamber holding a
solution containing a plurality of beads each of which has
biomolecular probes or protein immobilized thereon; a vibration
generator to vibrate the chamber; a long and narrow bead capturing
nozzle to capture the beads at a tip thereof; a suction pump
connected to the bead capturing nozzle; and an actuator to insert
the tip of the bead capturing nozzle into the solution in the
chamber as well as lift up the bead capturing nozzle, wherein an
opening formed at the tip of the bead capturing nozzle is smaller
than a diameter of each of the beads, and an outer diameter of the
tip of the bead capturing nozzle is larger than the diameter of
each of the beads.
18. The device for capturing beads according to claim 17, wherein
the beads have diameters of several tens of micrometers to several
millimeters.
Description
CLAIM OF PRIORITY
The present application claims priority from Japanese application
JP 2005-141569 filed on May 13, 2005, the content of which is
hereby incorporated by reference into this application.
FIELD OF THE INVENTION
The present invention relates to a device for capturing beads to
manipulate beads with biomolecular probes such as DNA, RNA, and
protein immobilized on their surface one by one and to a method and
apparatus for arraying beads to arrange the beads in a container by
manipulating a plurality of beads one by one.
BACKGROUND OF THE INVENTION
With the progress of the genome project, activities to understand
the cause of a disease and life phenomenon through understanding an
organism at its DNA level have become very active. For
understanding the life phenomenon and gene function, studies on
gene expression are effective. As a promising method to study this
gene expression, probe array, so-called DNA chip, in which a number
of DNA probes are immobilized on a solid surface such as slide
glass by sorting them out for every kind has come into use. The
method of producing DNA chips includes a method in which a
nucleotide oligomer with a designed sequence is synthesized base by
base in a large number of sectioned cells using a lithography
technology widely used in a photochemical reaction and a
semiconductor industry (Science 251, 767-773 (1991)) and a method
in which a plurality of kinds of DNA probes are spotted one by one
to each section (Science 270, 467-470 (1995); Nat. Biotechnol. 18,
438-441 (2000)).
When a DNA chip is produced, the DNA probes need to be immobilized
on an array piece by piece or the oligomers need to be synthesized
base by base in any of these methods, and its production requires
both time and manpower, leading to a high cost. In addition, since
the probes are immobilized by applying liquid droplets containing
them on a solid surface, there are problems that spot-to-spot
variation may result, changing a combination of kinds of the probes
is not easy, handling by a user is difficult, and so forth.
In order to solve the above problems, a probe array, that is, bead
array in which beads immobilized with DNA probes are prepared and a
plurality of kinds of these beads are lined up has been disclosed
(Clinical Chemistry 43, 1749-1756 (1997); Nucleic Acids Research
30, e87 (2002); Specification of U.S. Pat. No. 6,023,540). The
advantage of the probe array with beads lies in that a probe array
without variations in the probe density for each bead can be
produced because a method of probe immobilization with the use of a
chemical reaction in a solution can be employed.
In the DNA chip, the identification of a probe is performed by way
of the location of oligomer synthesis or the spot of each DNA
probe. In the probe array having beads immobilized with probes, the
identification of a probe is performed by way of beads colored
differently for each probe (Clinical Chemistry 43, 1749-1756
(1997); Specification of U.S. Pat. No. 6,023,540) or the order of
beads arrayed in a capillary (Nucleic Acids Research 30, e87
(2002)).
For the identification and quantitative analysis of a plurality of
kinds of DNA contained in an analyte sample with a DNA chip, the
sample is allowed to react with oligomers or DNAs immobilized on
the chip over half a day to a day. On the other hand, in a probe
array arranged with beads in a capillary, that is, bead array, an
analyte sample is forced to flow through the capillary. Since the
time required for gene examination with the bead array can be
shortened compared with a conventional method, the bead array is a
technique of measurement suitable for application at a clinical
site such as hospital. For example, the use of bead array can be
expected as a means for prompt detection of an infectious disease
requiring an urgent diagnosis and a foreign gene non-existent in
human and derived from the genome of a pathogenic microorganism in
bacteriological examination and the like.
For practical use of a bead array employing a method in which
probes are identified from the order of beads arranged in a
capillary (Nucleic Acids Research 30, e87 (2002)), it is essential
to establish a method for selecting arbitrary beads immobilized
with probes according to specific examination purpose and arraying
the beads as one desires, and several methods have been proposed
for this. For example, there are a method in which beads are poured
into a capillary by making use of a liquid flow while controlling
individual beads one by one (JP-A No. 243997/1999) and another
method in which only one bead is retained on a sheet provided with
a fine recess in which only one bead from among a plurality of the
beads introduced with a solvent can be put and the sheet is moved
to a position of a capillary or a slot provided on a flat plate
while retaining the bead, followed by arraying the bead one after
another (JP-A No. 346842/2000). However, these methods often fail
to incorporate beads because of the influence of air bubbles, and
therefore there was a problem in reliability and usability.
Hence, a method in which only one bead is captured at the suction
tip of a bead capturing nozzle from among a plurality of the beads
immobilized with the same probe with the use of the bead capturing
nozzle (JP No. 3593525; JP-A No. 17224/2005; Analytical Chemistry
75, 3250-3255(2003)). According to this method, beads can be
arrayed in the order as intended. In order to capture only one bead
at the suction tip of the bead capturing nozzle, beads additionally
attached to the side surface of the bead capturing nozzle due to
static electricity need to be removed. For this purpose, the
surface tension of the air-liquid interface that arises at the
border of the air and a solution is utilized as a means. The extra
beads attached to the sidewall of the bead capturing nozzle cannot
be passed through the interface and are retained on the solution
side of the interface when the bead capturing nozzle is withdrawn
from the solution into the air. Since the beads retained on the
interface cannot be dislocated into the air, the beads attached to
the sidewall of the bead capturing nozzle slide down along the
sidewall and left behind in the solution. As the result, only one
bead held via suction is captured by the bead capturing nozzle
after withdrawing the bead capturing nozzle into the air. It should
be noted that extra beads attached to the tip surface are taken out
into the air since the tip surface of the nozzle is not influenced
by the force from the air-liquid interface. To prevent this from
occurring, it is necessary not to provide any space at the tip
surface that allows bead attachment besides the suction portion,
and therefore it has been necessary to use the bead capturing
nozzle having an outer diameter approximately equal to that of the
bead.
SUMMARY OF THE INVENTION
Glass beads and plastic beads used for immobilization of
biomaterials had large variations in size, and even when a bead
capturing nozzle prepared by adjusting beforehand to the size of
the beads was used, two or more beads were often captured at the
tip surface of the bead capturing nozzle. On the other hand, when
beads having different diameters were arrayed on the same array by
a method in which a conventional bead capturing nozzle was used,
bead capturing nozzles suitable for each size of the beads to be
captured had to be prepared because the outer diameter of the bead
capturing nozzle was limited by the bead diameter.
The objects of the present invention are to provide a device for
capturing beads one by one that can reliably capture and manipulate
beads individually without depending too much on bead outer
diameter and an apparatus for arraying beads with the use of the
device for capturing beads one by one.
In the present invention, a bead holding plate having a plurality
of chambers each of which can hold a plurality of beads and a
solution, a vibration generator mounted with a stage where the bead
holding plate is arranged, and a bead capturing nozzle that is
connected to a suction pump and operated by aspirating its tip to
capture the bead are used.
The device for capturing beads one by one of the present invention
is provided with at least a chamber to hold the solution containing
the plurality of beads, the vibration generator to vibrate the
chamber, the long and narrow bead capturing nozzle to capture the
bead at its tip, the suction pump connected to the bead capturing
nozzle, and an actuator to insert the tip of the bead capturing
nozzle into the solution in the chamber as well as to lift it up.
An opening formed at the tip of the bead capturing nozzle is
smaller than the diameter of the beads and the outer diameter of
the tip of the bead capturing nozzle is larger than the diameter of
the beads. The vibration generator generates a vibration of a
frequency equal to or higher than 20 Hz. The amplitude of the
vibration is preferably equal to or larger than 0.1 mm.
The method for arraying beads according to the present invention
includes the steps of inserting the bead capturing nozzle, which
has the opening smaller than the diameter of the beads formed at
the tip and the outer diameter of the tip larger than the diameter
of the beads, into the solution containing a plurality of beads
immobilized with a biomolecular probe on the surface, exerting a
suction force on the tip of the bead capturing nozzle, vibrating
the chamber, withdrawing the bead capturing nozzle retaining one
bead at the tip via suction from the solution in the chamber, and
introducing the bead retained at the tip of the bead capturing
nozzle via suction into a bead array container.
The apparatus for arraying beads is provided with the stage to
retain a plurality of chambers to hold solutions each containing a
plurality of beads, the bead capturing nozzle having the opening
smaller than the diameter of the beads formed at the tip and the
outer diameter of the tip larger than the diameter of the beads,
the actuator to drive the bead capturing nozzle, a vacuum/pressure
unit to develop a negative or positive pressure at the tip of the
bead capturing nozzle, a vibration unit to vibrate the stage, a
holder for bead array container that retains a bead array container
to hold beads, and a control unit, where the control unit allows
the bead capturing nozzle to be inserted into the vibrating chamber
on the stage by controlling the actuator, the bead capturing nozzle
to be lifted up from the chamber by controlling the actuator in a
state that a single bead is captured at the tip of the bead
capturing nozzle by controlling the vacuum/pressure unit to develop
a negative pressure at the tip of the bead capturing nozzle, the
bead capturing nozzle capturing a single bead at the tip to be
moved to the position of the bead array container by controlling
the actuator, and the captured bead to be introduced into the bead
array container by controlling the vacuum/pressure unit to develop
a positive pressure at the tip.
According to the present invention, a single bead can be captured
from among the beads immobilized with biomolecules without fail,
and a bead array arranged with captured beads can be efficiently
produced at low cost.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram explaining a method for preparing beads and a
structure example of a bead holding plate;
FIG. 2 is a schematic diagram explaining a device for capturing
beads one by one according to the present invention;
FIGS. 3A to 3C are diagrams explaining a rocking vibration of the
bead holding plate;
FIGS. 4A to 4F are diagrams explaining a circular vibration of the
bead holding plate;
FIG. 5A is a schematic diagram showing a state just before a bead
capturing nozzle is inserted into a well;
FIG. 5B is a schematic diagram showing a state that the bead
capturing nozzle is inserted into the well;
FIG. 5C is a schematic diagram showing a state that a vibration is
applied to the well;
FIG. 5D is a schematic diagram showing a state of the moment when
the bead capturing nozzle is separated from a group of settling
beads;
FIG. 5E is a schematic diagram showing a state that the bead
capturing nozzle is withdrawn outside of the well;
FIG. 6 is a graph showing a relation between the number of captured
beads and vibration frequency;
FIGS. 7A to 7C are schematic diagrams explaining a set of
continuous microphotographs showing a process of bead capture,
where FIG. 7A is a microphotographic diagram showing a state that
the bead capturing nozzle is inserted into the beads, FIG. 7B is a
microphotographic diagram showing the moment when the bead
capturing nozzle is separated from the beads, and FIG. 7C is a
microphotographic diagram showing a state that the tip of the bead
capturing nozzle is withdrawn into the air;
FIGS. 8A to 8C are schematic diagrams explaining another set of
continuous microphotographs showing the process of bead capture,
where FIG. 8A is a microphotographic diagram showing a state that
the bead capturing nozzle is inserted into the beads, FIG. 8B is a
microphotographic diagram showing the moment when the bead
capturing nozzle is separated from the beads, and FIG. 8C is a
microphotographic diagram showing a state that the tip of the bead
capturing nozzle is withdrawn into the air;
FIG. 9A is a schematic diagram showing a structure example of an
apparatus for arraying beads according to the present
invention;
FIG. 9B is a schematic cross sectional view showing the structure
example of the apparatus for arraying beads according to the
present invention;
FIG. 10 is a schematic diagram showing a plurality of bead arrays
prepared by the use of the apparatus for arraying beads of the
present invention;
FIGS. 11A and 11B represent schematic diagrams showing a
hybridization experiment, where FIG. 11A is a diagram explaining
its method and FIG. 11B is a diagram explaining its result;
FIGS. 12A and 12B represent schematic diagrams showing a bead array
chip for arranging beads having different diameters in a plurality
of flow channels respectively, where FIG. 12A is a schematic
diagram of the chip before arraying the beads and FIG. 12B is a
schematic diagram of the chip after arraying the beads;
FIG. 13 is a schematic diagram showing a structure example of an
apparatus for arraying beads on a chip according to the present
invention; and
FIGS. 14A and 14B represent schematic diagrams of the bead
capturing nozzle.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, embodiments of the present invention are explained
with reference to the accompanying drawings.
First, the method for preparing beads immobilized with biomolecules
on their surface is explained with the use of FIG. 1. A bead
holding plate 2 having the m.times.n number of wells 1, groups of
beads, and a plurality of kinds of biomolecular probes such as DNA,
RNA, and protein that modify beads 3 are prepared. The wells 1 on
the prepared bead holding plate 2 are spaced uniformly not only in
the x direction at intervals of a first center distance but also in
the y direction perpendicular to the x direction at intervals of a
second center distance. The well 1 has a circular upper opening and
a central axis parallel to the z direction, and is either in a
cylindrical or cone shape having a bottom portion. As the bead
holding plate 2 having such a plurality of the wells 1, for
example, a commercially available 384-well microtiter plate can be
used. Preferably the beads 3 to be used are 10 micrometers or
larger in diameter and in a spherical shape when a commercially
available capillary is used for capturing beads, while beads of
several micrometers in diameter can also be used when a nozzle
exclusively for capturing beads that is micro-fabricated by
semiconductor etching technology is used.
The beads 3 prepared in a several mg unit are distributed to each
well on the bead holding plate 2 from a bead container 4 using a
spatula. Then, different kinds of probes are introduced onto the
beads present in a row of the wells 1 as a unit or in each well 1,
and the probes are immobilized on the surfaces of all beads. In
this way, the bead holding plate 2 that retains a plurality of
probe-immobilized beads 5 in which each kind of the probes is
correlated with the position of the wells 1 can be prepared. In the
present example, n kinds of biomolecular probes are prepared, and
No. 1 biomolecular probe is introduced into m wells in the first
row, No. 2 biomolecular probe into m wells in the second row, and
so on, and No. n biomolecular probe is introduced into m wells in
the n-th row, thereby immobilizing the probes on the beads 3 held
in each well 1. When the probes immobilized on the beads 3 on the
bead holding plate 2 are relatively chemically stable biomolecules
such as DNA, the prepared bead holding plate 2 can be stored in a
desiccator or a refrigerator, thus allowing its preparation to be
conducted in bulk. The bead holding plate 2 in which a solution
such as pure water is introduced into each well 1 is mounted on a
device for capturing beads one by one and a stage for bead holding
plate 6 of an apparatus for arraying beads as described later.
FIG. 2 is a schematic diagram explaining an example of the device
for capturing beads one by one according to the present invention.
This device for capturing beads one by one is provided with a
vibration generator 7 mounted with the stage for bead holding plate
6 where the bead holding plate 2 is arranged, a bead capturing
nozzle 8, a suction pump 9, a first electric actuator 12 to drive
the bead capturing nozzle 8 in the z direction, and a second
electric actuator 13 to drive the bead capturing nozzle 8 in the xy
directions. The bead capturing nozzle 8 is retained by a bead
capturing nozzle retaining member 11 fixed on the electric actuator
12 and connected to the suction pump via a tube 10. The vibration
generator 7 can be varied in vibration amplitude and vibration
frequency. The structure of the apparatus for arraying beads
provided with the device for capturing beads one by one is
described later.
The vibration generator 7 vibrates the bead holding plate 2 by
moving the stage for bead holding plate 6 in the direction of the x
axis or the y axis as shown in FIGS. 3A to 3C. FIGS. 3A to 3C show
a case where vibration is generated in a reciprocating motion in
the direction of the x axis. The vibration generator 7 may be
driven in a circular motion of the bead holding plate 2 centered at
a certain base point as shown in FIGS. 4A to 4F. Further, the
amplitude of the vibration may be given in the direction of the z
axis. However, when the bead holding plate 2 is vibrated in the
direction of the z axis, a solution contained in the well 1 tend to
overflow, and there is a risk that the bead capturing nozzle 8 and
the bottom of the well 1 collide with each other, therefore
rendering it undesirable in view of safe operation of the
apparatus. Further, the vibration may also be given to the bead
capturing nozzle 8. However, accuracy of positioning by the bead
capturing nozzle retaining member 11 and reproducibility of
positioning of the tip of the bead capturing nozzle 8 deteriorate,
rendering it undesirable in view of safe operation of the
apparatus.
FIGS. 14A and 14B show the shapes of the bead capturing nozzle 8
schematically. FIG. 14A shows a typical shape of the bead capturing
nozzle 8 having uniform inner and outer diameters from the tip to
the end, and the bead capturing nozzle of this structure can make
use of a commercially available capillary. FIG. 14B shows a shape
of the bead capturing nozzle with its tip tapered, and the bead
capturing nozzle of this structure can make use of a capillary for
wire bonding that is used in semiconductor manufacturing. In
addition, these nozzles can be fabricated by a semiconductor
etching technique. The material used for the nozzle includes
stainless steel, glass, ceramic, ruby, silicon, and the like. In
the example below, a bead capturing nozzle having a flat tip shape
as shown in FIG. 14A was used.
FIGS. 5A to 5E are schematic cross sectional views showing the
steps of picking up only one bead 3 from a plurality of beads held
in the well 1 together with a solution 14 with the use of the
device for capturing beads one by one shown in FIG. 2. The solution
14 here is pure water.
FIG. 5A depicts a state that the bead capturing nozzle 8 is moved
by the second electric actuator 13 such that the opening of the
well 1 retaining target beads 3 is set at a position opposite to
the opening of the bead capturing nozzle 8 in the z direction. The
figure shows a state that the bead capturing nozzle 8 is just about
to be inserted into the target well 1. At this time, the suction
pump 9 connected to the bead capturing nozzle 8 is driven, and a
negative pressure is created at the tip of the bead capturing
nozzle 8. Alternatively, the suction pump 9 is kept driven at all
times, an electromagnetic valve 15 is inserted between the bead
capturing nozzle 8 and the suction pump 9 in advance, and the
electromagnetic valve 15 may be controlled by switching.
FIG. 5B depicts a state that the bead capturing nozzle retaining
member 11 descends in the z direction by the control of the first
electric actuator 12 and the lower tip of the bead capturing nozzle
8 in which a negative pressure is created is inserted into the
inside of the well 1. The distance between the tip surface of the
bead capturing nozzle 8 and the bottom of the well 1 is adjusted
appropriately so as to make contact with each other in the inserted
state. This is because a decrease of efficiency of bead capturing
caused by inaccessibility of the tip of the bead capturing nozzle 8
to the bead 3 is prevented when the content of the beads 3 is
low.
FIG. 5C depicts a state that the vibration generator 7 is driven
and the bead holding plate 2 is vibrated at a predetermined
frequency and predetermined amplitude. The figure depicts a state
that the bead holding plate 2 is vibrated only in the direction of
the x axis. The extent of the vibration to be given is sufficient
if the surface of the solution 14 is shaken. It is unnecessary for
the beads 3 to be stirred. In other words, it is a state that a
certain vibration is given to the entire group of the precipitated
beads. The upper and lower limits of the frequency and amplitude
provided here are described later based on the experimental result
in FIG. 6. Although the vibration generator 7 is driven after the
bead capturing nozzle 8 is inserted in FIG. 5C, the vibration
generator 7 may be kept driven from the time point in FIG. 5A.
FIG. 5D depicts the right moment at which the bead capturing nozzle
8 leaves the group of beads settling at the bottom of the well 1.
At this time, only one bead 3 is captured at the tip of the nozzle.
When the amplitude is generated at a frequency below the lower
limit described later, a plurality of the beads 3 are attached to
the tip surface. In addition, the beads 3 are also adsorbed on the
sidewall of the bead capturing nozzle 8.
FIG. 5E depicts a state that the bead capturing nozzle 8 is moved
to the z direction farther than the state in FIG. 5D where the tip
of the bead capturing nozzle 8 passes through the interface between
the solution 14 and the air and is withdrawn completely into the
air.
According to the steps explained by FIGS. 5A to 5E, one bead 3 is
retained at the tip of the bead capturing nozzle 8 without
fail.
First Embodiment
FIG. 6 depicts the relation between the vibration frequency and the
number of captured beads 3, showing how the beads were captured
when the frequency and amplitude for vibration in the direction of
the x axis in FIG. 5C were varied. In this experiment, the beads
having a diameter of 0.1 mm and the bead capturing nozzle 8 having
an inner diameter and outer diameter of 0.05 mm and 0.4 mm,
respectively, were used. For the wells 1, wells of a 384-well
microtiter plate having an opening diameter of 3.5 mm were used.
Three milligrams of the beads 3 were introduced into each well 1
using a spatula, followed by addition of 60 microliters of pure
water 14. The operation of bead capture was repeated ten times for
every frequency, and the average number of the captured beads was
determined and plotted.
FIG. 6 shows that a smaller amplitude is sufficient as the
frequency becomes higher. However, when the amplitude was smaller
than 0.1 mm, it was not possible to capture only one bead 3 even
when the frequency was increased as shown in FIG. 6. Hence, it can
be said that a frequency equal to or higher than 20 Hz and
amplitude equal to or larger than 0.1 mm are lower limits required
for capturing a single bead. Further, it is shown that a single
bead could be captured without fail by applying a frequency equal
to or higher than 20 Hz in the range of the amplitude equal to or
larger than 0.4 mm. This result indicates that only one bead 3 can
be captured at an approximate frequency of 20 Hz when an amplitude
equal to or larger than the outer diameter of the nozzle is
provided.
When the vibration frequency was equal to or higher than 1,000 Hz,
it was found difficult to capture the bead 3 at any amplitude.
However, it is expected to be able to capture the bead even at a
frequency equal to or higher than 1,000 Hz when changes such as
decreasing flow resistance in the suction path and enhancing flow
rate by means of increasing the inner diameter and further the
inner diameter other than the tip are carried out in the inner
structure of the bead capturing nozzle 8. Although the amplitude
was changed only up to 3 mm because of the size of the well 1, it
is evident from this result that the bead can be captured even when
the amplitude is larger than 3 mm.
Although it was possible to control the beads in a range of
amplitude from 0.1 mm to 3 mm, a load is applied to the bead
capturing nozzle 8 when a large amplitude is given while the bead
capturing nozzle is inserted into the group of beads, and thus
there is a possibility that the tip may be distorted or may further
be broken. Therefore, it is better to set the amplitude small.
FIGS. 7 and 8 are images showing bead capture by the use of the
device for capturing beads one by one of the present invention.
FIGS. 7A to 7C represent part of continuous microphotographic
diagrams showing a process of the bead capture when vibration is
provided by the vibration generator 7. The condition of the
vibration consisted of a frequency of 20 Hz and an amplitude of 0.4
mm. FIG. 7A is a microphotographic diagram showing a state that the
bead capturing nozzle 8 in which a negative pressure was created
was inserted into a group of beads 16 in the well 1. FIG. 7B is a
microphotographic diagram showing the moment when the bead
capturing nozzle 8 was separated from the group of beads 16
settling at the bottom of the well 1. It is seen that only one bead
3 was captured exclusively at the tip of the bead capturing nozzle
8 at this time point. FIG. 7C is a microphotographic diagram
showing a state that the tip of the bead capturing nozzle 8 was
withdrawn into the air. It is seen that only one bead 3 was
captured at the tip of the bead capturing nozzle 8 even after
passing through the air-liquid interface.
FIGS. 8A to 8C represent part of continuous microphotographic
diagrams showing a result of the bead capture when the vibration
frequency provided by the vibration generator 7 was lower than 20
Hz. The condition of the vibration consisted of a frequency of 10
Hz and an amplitude of 0.3 mm. FIG. 8A is a microphotographic
diagram showing a state that the bead capturing nozzle 8 in which a
negative pressure was formed was inserted into the group of beads
16 in the well 1. FIG. 8B is a microphotographic diagram showing
the moment when the bead capturing nozzle 8 was separated from the
group of beads 16 settling at the bottom of the well 1. It is seen
from FIG. 8B that a plurality of the beads were attached to the
side surface and portions of the tip surface other than the suction
portion of the bead capturing nozzle 8 in the solution 14. FIG. 8C
is a microphotographic diagram showing a state that the tip of the
bead capturing nozzle 8 was withdrawn into the air. It is seen that
the beads on the side surface were removed by the surface tension
of an air-liquid interface 17 as the result of withdrawing the bead
capturing nozzle 8 into the air, while extra beads 3 remained
sticking to the tip surface.
The results in FIGS. 7 and 8 indicate that only one bead 3 can be
adsorbed and captured at the tip of the bead capturing nozzle 8
from the group of beads 16 by applying an appropriate vibration to
a chamber, i.e. the well 1, containing the beads and the
solution.
Second Embodiment
FIGS. 9A and 9B are schematic diagrams showing a structure of an
apparatus for arraying beads provided with the device for capturing
beads one by one according to the present invention, where FIG. 9A
is a perspective view and FIG. 9B is a cross sectional view. The
vibration generator 7 and the stage for bead holding plate 6
constituting the device for capturing beads one by one of the
present invention were placed on a plate-like base 18, and the bead
capturing nozzles 8 retained by the bead capturing nozzle retaining
member 11 was arranged above the stage 6. The bead capturing nozzle
8 was connected to a vacuum/pressure pump 23. The bead capturing
nozzle retaining member 11 was driven by the first electric
actuator 12 and the second electric actuator 13. The base 18 was
provided with through-holes for movement assistance 19, and a first
image sensor 20, a second image sensor 21, and a suction pump for
introducing beads 22 were arranged. The numeral 24 represents a
control computer.
The details of the device for capturing beads one by one have been
explained in FIGS. 1 to 4. In the present embodiment, a structure
having an arrangement of a plurality of the bead capturing nozzles
8 was employed, and n pieces of the bead capturing nozzles 8 were
provided in parallel with the direction of the y axis at the same
spacing as that for the wells 1 on the bead holding plate 2. The
number of the bead capturing nozzles 8 was five in the illustrated
example. When a microtiter plate having the wells 1 of a diameter
of 3.5 mm and a depth of 9.6 mm arranged in a total of 384, 24
(m=24) in the direction of the x axis and 16 in the direction of
the y axis (n=16), is used, it is desirable to arrange 16 bead
capturing nozzles 8 in parallel with the direction of the y axis.
The positions of the tips of the bead capturing nozzles 8 were
aligned in the direction of the z axis and these were firmly
retained by the bead capturing nozzle retaining member 11 so as to
be handled as a group. The bead capturing nozzles were connected to
the vacuum/pressure pump 23 capable of aspirating as well as
pressurizing. The suction and pressurization can be arbitrarily
selected by switching a valve.
The through-holes for movement assistance 19 were provided in n
pieces in parallel with and at the same spacing as that for the
wells 1 arranged in the direction of the y axis of the bead holding
plate 2. The cross section of the through-hole for movement
assistance 19 is circular and has a central axis parallel to the z
axis. Since the through-holes for movement assistance 19 were
provided to support insertion of the beads 3 adsorbed on the tip
openings of the bead capturing nozzles 8 into capillaries for bead
array 25 while guiding a plurality of the bead capturing nozzles 8
and the capillaries for bead array 25, the inner diameter of the
through-holes for movement assistance 19 is designed such that the
capillary for bead array 25 and the bead capturing nozzle 8 can
move safely. For example, when the bead capturing nozzle 8 has an
outer diameter of 0.4 mm and an inner diameter of 0.05 mm, the
inner diameter of the through-hole for movement assistance 19 is
set to 0.5 mm.
The first image sensor 20 was placed at a location parallel and
adjacent to the through-holes for movement assistance 19. This
image sensor 20 was used to confirm whether the beads 3 were
captured at the tips of the bead capturing nozzles 8 one by one
before the bead capturing nozzles 8 were introduced into the
through-holes for movement assistance 19. When it was found from an
output of the first image sensor 20 that there was any bead
capturing nozzle 8 on which the bead 3 was not captured, the
operation of bead capture by the bead capturing nozzle 8 was
repeated.
The second image sensor 21 was placed at a location opposite to the
first image sensor 20 and on the undersurface side of the base 18
and used to confirm whether the beads 3 were introduced into the
capillaries for bead array 25.
The capillaries for bead array 25 were provided in n pieces in
parallel with the direction of the y axis at the same spacing as
that for the wells 1 on the bead holding plate 2 and also
corresponded to the bead capturing nozzles 8. Although the number
of the capillaries for bead array 25 was five in the illustrated
example, sixteen of these are arranged in parallel with the
direction of the y axis in the structure of the above example with
the titer plate. One end of the capillary for bead array 25 had an
opening, and the other end was connected to the suction pump for
introducing beads 22 via a tubeland aspirated. It is desirable that
the end portion of the capillary for bead array 25 and the tube are
connected via a socket 26. Here, the socket 26 had an inner
diameter smaller than the outer diameter of the beads 3 so as to
prevent the passage of the beads 3.
With the use of the apparatus for arraying beads, the beads 3 were
aspirated into the capillaries for bead array 25 one by one, and a
plurality of the beads 3 were arrayed therein while keeping the
order of the beads that had been aspirated.
When the diameter of the bead 3 is R, the inner diameter ID of the
capillary for bead array 25 satisfies the relation of
R<ID<2R. When the diameter of the bead 3 is 0.1 mm, the inner
diameter and outer diameter of the capillary for bead array 25 may
be set to 0.15 mm and 0.38 mm, respectively.
It is advisable that the end portions on the opening side of the
capillaries for bead array 25 are retained by a holder 27 in a
firmly fixed manner so that the tip portions of the capillaries for
bead array 25 may be aligned in the direction of the z axis and
they are handled as a group. Of course, it is advisable that the
capillaries for bead array 25 may be released from being retained
by the holder 27 and may be treated as individual capillaries for
bead array 25 after necessary beads 3 have been inserted into
them.
After the beads 3 had been adsorbed to the tip openings of the bead
capturing nozzles 8 via the operation process for the device for
capturing beads one by one explained by the use of FIGS. 1 and 2,
the bead capturing nozzles 8 were moved by the first electric
actuator 12 and the second electric actuator 13, and the tip
openings of the bead capturing nozzles 8 and the openings of the
capillaries for bead array 25 were allowed to be opposite to each
other within the through-holes for movement assistance 19. Then,
the tubes connected to the bead capturing nozzle 8 were pressurized
to release the beads adsorbed to the tip openings, thereby allowing
the beads to be transferred into the capillaries for bead array 25.
At this time, the beads 3 were effectively introduced into the
capillaries for bead array 25 by aspirating the inside of the
capillaries for bead array 25.
FIG. 10 is a schematic cross sectional view of an example of a
plurality of the capillaries for bead array 25 obtained by the use
of the bead holding plate 2 having m.times.n wells 1 according to
the present embodiment. At this stage, narrow hollow tubes having
an outer diameter approximately equal to the inner diameter of the
capillary were inserted into both ends of the capillary so that the
beads 3 introduced into the capillary might not drop out and be
kept in an orderly arrayed state, thereby preventing any movement
of the beads 3. However, the narrow hollow tube to be inserted into
the left side is omitted in the illustrated example. This is
because it is necessary to introduce a sample into the capillary
for bead array 25.
An example of the use of the capillaries for bead array in which
fluorescently labeled specific target DNAs were hybridized to a DNA
probe array that was prepared by arraying 24 kinds of
DNA-immobilized beads attached with DNA probes one by one in an
arbitrary order in the capillaries for bead array according to the
example of the apparatus for arraying beads in FIG. 9 is explained
with reference to FIGS. 11A and 11B.
In FIGS. 11A and 11B, it was determined whether target DNAs bound
to their corresponding probe DNAs as intended. For this purpose, 24
kinds of 18-mer synthetic oligonucleotides having each different
sequence modified at the 5'-end by a thiol group were prepared as
probes. Among the 24 kinds of the probes, the capillary for bead
array 25 provided with both of a bead immobilized with a single
stranded DNA probe 28 having Sequence 1 and a bead immobilized with
a single stranded DNA probe 29 having Sequence 2 was allowed to
come in contact with a flow of a sample containing a single
stranded target DNA 30 having Sequence 3 complementary to Sequence
1 and labeled with Cy3 and a single stranded target DNA 30 having
Sequence 4 complementary to Sequence 2 and labeled with
TexasRed.
TABLE-US-00001 5'-thiol-ATCTGACT . . . GCTCCTC-3' (Sequence 1)
5'-thiol-CTACCTGC . . . CTGGACG-3' (Sequence 2) 5'-Cy3-GAGGAGCC . .
. GTCAGAT-3' (Sequence 3) 5'-TexasRed-CGTCCAGG . . . CAGGTAG-3'
(Sequence 4)
A solution of 20 mM phosphate buffer (pH 7.0) 32 containing the
single stranded target DNA 30 and the single stranded target DNA 31
at a concentration of 1 .mu.M respectively was flown into the
capillary for bead array 25 with the prepared DNA probe array and
subjected to hybridization at 45 degrees c. Feeding of the solution
into the capillary was carried out with a syringe pump. After the
reaction, the residual target DNA not involved in the hybridization
reaction was washed successively with the 20 mM phosphate buffer
(pH 7.0) solution 32 and pure water, followed by drying. Then, each
bead in the capillary for bead array was observed by a fluorescent
microscope 33 with a mercury lamp as light source using in turn a
long-pass filter for Cy3 and a long-pass filter for TexasRed that
were centered around emission wavelengths of Cy3 and TexasRed,
respectively.
As the result, it was observed as shown in FIG. 11B that a specific
bead generated fluorescence of Cy3 34 and another specific bead
generated fluorescence of TexasRed 35, respectively, among the
arrayed beads. This indicated that the single stranded target DNAs
30 and 31 specifically hybridized to the single stranded DNA probes
28 and 29, respectively. Thus, it was confirmed that, with the use
of this apparatus for arraying beads, a DNA probe array can be
prepared in the capillary for bead array 25 in an arbitrary linear
arrangement without exerting an influence on probes.
Third Embodiment
Glass beads and plastic beads immobilized with biomolecular probes
often exhibit variations in size. Further, the size of beads to be
used differs depending on the difference in assay systems for
genetic tasting. Here, a model experiment in which various beads
having diameters larger than the diameter of the tip opening of the
bead capturing nozzle 8 but equal to or smaller than its outer
diameter were manipulated by one kind of bead capturing nozzle 8 is
explained.
In the experiment, beads of 0.05 mm 36, beads of 0.1 mm 37, beads
of 0.3 mm 38, beads of 0.5 mm 39, and beads of 1 mm 39 were arrayed
on a flow channel chip 41 having five different flow channel cross
sections depending on each size of the beads using the apparatus
for arraying beads shown in FIG. 13 in which the device for
capturing beads one by one was mounted with the bead capturing
nozzle 8 having an outer diameter of 1 mm and an inner diameter of
0.04 mm. FIG. 12A is a schematic diagram of the flow channel chip
before arraying the beads, and FIG. 12B is a schematic diagram of
the flow channel chip after arraying the beads.
The cross sectional shapes of the five flow channels on the chip
may be either square or circular. In the present embodiment, a chip
having flow channels in square cross section was used.
Specifically, a chip 41 having five flow channels consisting of a
square flow channel having a side of 0.07 mm 43, a square flow
channel having a side of 0.13 mm 44, a square flow channel having a
side of 0.35 mm 45, a square flow channel having a side of 0.6 mm
46, and a square flow channel having a side of 1.2 mm 47 was used.
In each flow channel, a weir 48 was provided as a stopper for the
beads, and the beads were introduced through a circular hole
connected to the flow channel called a bead introduction inlet 49.
The numeral 50 represents an opening for piping. When a bead array
chip 42 was prepared, this opening was connected to a tube, and
further to a suction pump, thereby aspirating the inside of the
flow channel. That is, the beads can be introduced efficiently into
the flow channel by the suction effect from the side of the opening
for piping 50.
FIG. 13 is a schematic diagram showing a structure of an apparatus
for arraying beads on a chip. Although its basic structure is
similar to the apparatus shown in FIG. 9, it is structured such
that the beads captured by the device for capturing beads one by
one are introduced directly into bead introduction inlets 49
located at one ends of the flow channels of the flow channel chip
41 and further the side of the openings for piping 50 is connected
to the suction pump for introducing beads 22 via tubes.
Five bead capturing nozzles 8 having an outer diameter of 1 mm and
an inner diameter of 0.04 mm were arranged. For the bead holding
plate 2, beads having the same diameter were placed in a row of the
four rows in the direction of the x axis in FIG. 13, and beads
having different diameters were placed in the five rows in the
direction of the y axis in FIG. 13, respectively. One piece of the
beads was taken out from each well in the direction of the m row
one after another by the bead capturing nozzle 8 and introduced
into the flow channel chip 41 having the five flow channels. The
vibration condition of the device for capturing beads one by one
was set to a vibration frequency of 20 Hz and amplitude of 1
mm.
A schematic diagram of a two-dimensional bead array chip 42 after
arraying is shown in FIG. 12B. The beads having the diameter of
0.05 mm 36, the beads having the diameter of 0.1 mm 37, the beads
having the diameter of 0.3 mm 38, the beads having the diameter of
0.5 mm 39, and the beads having the diameter of 1 mm 40 were
arrayed in the square flow channel having the side of 0.07 mm 43,
the square flow channel having the side of 0.13 mm 44, the square
flow channel having the side of 0.35 mm 45, the square flow channel
having the side of 0.6 mm 46, and the square flow channel having
the side of 1.2 mm 47, respectively. The number of arrayed beads
was the same among these channels, which indicates that the beads
were captured and introduced one by one.
SEQUENCE LISTINGS
1
4 1 18 DNA Artificial Sequence location of a fluorophore-tag 1;
(8)...(11) DNA primer incorporating a fluorophore-tag and n is a,
c, g or t 1 atctgactnn ngctcctc 18 2 18 DNA Artificial Sequence
location of a fluorophore-tag 1; (8)...(11) DNA primer
incorporating a fluorophore-tag and n is a, c, g or t 2 ctacctgcnn
nctggacg 18 3 18 DNA Artificial Sequence location of a
fluorophore-tag 1; (8)...(11) DNA primer incorporating a
fluorophore-tag and n is a, c, g or t 3 gaggagccnn ngtcagat 18 4 18
DNA Artificial Sequence location of a fluorophore-tag 1; (8)...(11)
DNA primer incorporating a fluorophore-tag and n is a, c, g or t 4
cgtccaggnn ncaggtag 18
* * * * *